Vegetable oils are increasingly important economically because they are widely used in human and animal diets and in many industrial applications. However, the fatty acid compositions of these oils are often not optimal for many of these uses. Because specialty oils with particular fatty acid composition are needed for both nutritional and industrial purposes, there is considerable interest in modifying oil composition by plant breeding and/or by new molecular tools of plant biotechnology.
The specific performance and health attributes of edible oils are determined largely by their fatty acid composition. Most vegetable oils derived from commercial plant varieties are composed primarily of palmitic (C16:0), stearic (C18:0), oleic (C18:1), linoleic (C18:2) and linolenic (C18:3) acids. Palmitic and stearic acids are, respectively, 16 and 18 carbon-long, saturated fatty acids. Oleic, linoleic, and linolenic acids are 18-carbon-long, unsaturated fatty acids containing one, two, and three double bonds, respectively. Oleic acid is referred to as a mono-unsaturated fatty acid, while linoleic and linolenic acids are referred to as poly-unsaturated fatty acids.
Brassica species like Brassica napus (B. napus) and Brassica rapa (B. rapa) constitute the third most important source of vegetable oil in the world. In Canada, plant scientists focused their efforts on creating so-called “double-low” varieties which were low in erucic acid in the seed oil and low in glucosinolates in the solid meal remaining after oil extraction (i.e., an erucic acid content of less than 2.0 percent by weight based upon the total fatty acid content (called herein after wt %), and a glucosinolate content of less than 30 micromoles per gram of the oil-free meal). These higher quality forms of rape developed in Canada are known as canola.
Oil extracted from natural and previously commercially useful varieties of canola contains a relatively high (8%-10%) alpha-linolenic acid content (C18:3) (US Patent Application 20080034457). Higher values of e.g. 11% have also been reported (http://www.canolacouncil.org/canola_oil_properties_and_uses.aspx). This trienoic fatty acid is unstable and easily oxidized during cooking, which in turn creates off-flavors of the oil (Gailliard, 1980, Vol. 4, pp. 85-116 In: Stumpf, P. K., ed., The Biochemistry of Plants, Academic Press, New York). It also develops off odors and rancid flavors during storage (Hawrysh, 1990, Stability of canola oil, Chapter 7, pp. 99-122 In: F. Shahidi, ed. Canola and Rapeseed: Production, Chemistry, Nutrition, and Processing Technology, Van Nostrand Reinhold, N.Y.). Both flavor and nutritional quality of the oil is improved by reducing the C18:3 levels in favor of C18:2 (Diepenbrock and Wilson, Crop Sci 27:75-77, 1987)
It is known that reducing the alpha-linolenic acid content level by hydrogenation increases the oxidative stability of the oil. Unfortunately, chemical hydrogenation leads to the formation of trans-fatty acids, which have been linked to elevated levels of low-density lipoprotein cholesterol (LDL or “bad” cholesterol) in the blood, and consequently, to an increased risk of coronary heart disease.
Another strategy to improve oil quality is by breeding for low linolenic varieties, which is particularly challenging since C18:3 content is a multi-gene trait and inherited in a recessive manner with a relatively low heritability (WO04072259). Burns et al. (Heredity 90:39-48, 2003) identified five qualitative trait loci (QTL) associated with C18:3 content in B. napus, of which three with positive effect located on N6, N7 and N18, and two with negative effect on N7 and N11. Genetic analysis of a population derived from the cross between “Stellar” (having a low C18:3 content (3%)) and “Drakkar' (having a “conventional” C18:3 level (9-10%)) indicated that the low C18:3 trait was controlled by two major loci with additive effects designated L1 and L2 (Jourdren et al., Euphytica 90:351-357, 1996). These two major loci controlling C18:3 content were found to correspond to two FAD3 (fatty acid desaturase 3) genes; one located on A genome (originating from Brassica rapa) on N4 and the other on the C genome (originating from Brassica oleracea) on N14 (Jourdren et al., Theor. Appl. Genet. 93:512-518, 1996; Barret et al., GCIRC, 1999).
Canola varieties with mutations in the FAD3 gene have been described in the art. For example, WO06/034059 describes that two canola varieties with reduced linolenic acid content, IMC01 and IMC02 (originally disclosed in U.S. Pat. No. 5,750,827 and US patent application 20080034457, respectively) are thought to have mutations in FAD3 genes. WO04/072259 discloses a FAD3 allele (of the C genome) with a single nucleotide substitution in a 5′ splice site from a mutant canola line DMS 100 with a linolenic acid content of about 3%. WO01/25453 describes new FAD3 variants with multiple amino acid substitutions, one of which is also present in “Stellar”, from the low linolenic “Apollo” variety. In US patent application 20040083503 a non-functional FAD3 mutant is disclosed with an amino acid substitution in a conserved domain. However, the linolenic acid phenotype of canola plants comprising such mutant FAD3 alleles can be highly variable depending on the genetic background.
Therefore, despite the fact that sequences of various FAD3 alleles are available in the art, a need remains for alternative methods (especially non-transgenic methods) for stably reducing the amount of alpha-linolenic acid in seed, without having a negative effect on the plants growth and development. The inventions described hereinafter in the different embodiments, examples and claims provide methods and means for developing crop plants which produce seed oil that is low in C18:3 content.